Optical imaging is an evolving clinical imaging modality that uses penetrating light rays to create images. Light in the red and near-infrared (NIR) range (600-1200 nm) is used to maximize tissue penetration and minimize absorption from natural biological absorbers such as hemoglobin and water. (Wyatt, Phil. Trans. R. Soc. London B 352:701-706, 1997; Tromberg, et al., Phil. Trans. R. Soc. London B 352:661-667, 1997).
Besides being non-invasive, optical imaging methods offer a number of advantages over other imaging methods: they provide generally high sensitivity, do not require exposure of test subjects or lab personnel to ionizing radiation, can allow for simultaneous use of multiple, distinguishable probes (important in molecular imaging), and offer high temporal and spatial resolution (important in functional imaging and in vivo microscopy, respectively).
In fluorescence imaging, filtered light or a laser with a defined bandwidth is used as a source of excitation light. The excitation light travels through body tissues. When it encounters a reporter molecule (i.e., contrast agent or imaging has detectably different properties from the excitation light. The resulting emitted light then can be used to construct an image.
Most optical imaging techniques have relied on the use of organic and inorganic fluorescent molecules as the reporter molecule.
Fluorescent dyes are generally known and used for fluorescence labeling and detection of various biological and non-biological materials by procedures such as fluorescence microscopy, fluorescence immunoassay and flow cytometry. A typical method for labeling such materials with fluorescent dyes is to create a fluorescent complex by means of bonding between suitable groups on the dye molecule and compatible groups on the material to be labeled. In this way, materials such as cells, tissues, amino acids, proteins, antibodies, drugs, hormones, nucleotides, nucleic acids, lipids and polysaccharides and the like may be chemically labeled and detected or quantified, or may be used as fluorescent probes which can bind specifically to target materials and detected by fluorescence detection methods. Brightly fluorescent dyes permit detection or location of the attached materials with great sensitivity.
Certain carbocyanine or polymethine dyes have demonstrated utility as labeling reagents for a variety of biological applications, e.g. U.S. Pat. No. 4,981,977 to Southwick, et al. (1991); U.S. Pat. No. 5,268,486 to Waggoner et al. (1993); U.S. Pat. No. 5,569,587 to Waggoner (1996); U.S. Pat. No. 5,569,766 to Waggoner et al. (1996); U.S. Pat. No. 5,486,616 to Waggoner et al. (1996); U.S. Pat. No. 5,627,027 to Waggoner (1997); U.S. Pat. No. 5,808,044 to Brush, et al. (1998); U.S. Pat. No. 5,877,310 to Reddington, et al. (1999); U.S. Pat. No.6,002,003 to Shen, et al. (1999); U.S. Pat. No. 6,004,536 to Leung et al. (1999); U.S. Pat. No. 6,008,373 to Waggoner, et al. (1999); U.S. Pat No. 6,043,025 to Minden, et al. (2000); U.S. Pat. No. 6,127,134 to Minden, et al. (2000); U.S. Pat. No. 6,130,094 to Waggoner, et al. (2000); U.S. Pat. No. 6,133,445 to Waggoner, et al. (2000); also WO 97/40104, WO 99/51702, WO 01/21624, and EP 1 065 250 A1; and Tetrahedron Letters 41, 9185-88 (2000); all of the above incorporated by reference.
Comprehensive reviews regarding polymethine dyes have been by written by L. G. S. Brooker, “The Theory of the Photographic Process” Mees Ed., Macmillan, New York, (1942), p. 987 and (1966), p. 198; Frances M. Hamer, in “The Chemistry of Heterocyclic Compounds”, Vol 18, “The Cyanine Dyes and Related Compounds”, Weissberger, Ed, Wiley Interscience, New York, (1964); G. E. Ficken, “The Chemistry of Synthetic Dyes”, Vol 4, K. Venkataraman Ed., Academic Press, New York, (1971), p. 211; A. I. Kiprianov, Usp. Khim., 29, 1336, (1960), 35, 361 (1966), 40, 594 (1971); D. W. Heseltine, “The Theory of the Photographic Process”, 4.sup.th edition, James Ed., Macmillan, New York, (1977), chapter 8, “Sensitising and Desensitising Dyes”; S. Daehne, Phot. Sci. Eng., 12, 219 (1979); D. J. Fry, “Rodd's Chemistry of Carbon Compounds”, “Cyanine Dyes and Related Compounds”, Vol. IVb, chapter 15, p. 369 Elsevier, Amsterdam, (1977); Supplement to Vol. IVb, 2.sup.nd Edition (1985), p. 267; H. Zollinger, “Color Chemistry”, VCH, Weinheim (1987), chapters 3 and 14; D. M. Stunner, “The Chemistry of Heterocyclic Compounds”, “Special Topics in Heterocyclic Chemistry”, chapter VIII, “Synthesis and Properties of Cyanine and Related Dyes”, Weissberger Ed., Wiley, New York, (1977); “The Kirk-Othmer Encyclopaedia of Chemical Technology” Vol 7, p. 782, “Cyanine Dyes”, Wiley, New-York, (1993).
To be useful as a label, a dye has to be provided with a suitable side chain containing a functional group. The method and site of introduction of a side chain containing a functional group into the structure for the purpose of conjugation, or binding to another molecule, represents the innovative step in the inventions concerning the use of the dye as a labeling reagent. Typically, only one such functionalized side arm is used in order to avoid cross-linking or purification problems. One aspect in the design of polymethine labeling reagents has been to attach the functionalized side arm to one of the heterocyclic nuclei of the dye, formula (1):Z1-PML-Z2  (1)See, for instance: J. S. Lindsey, P. A. Brown, and D. A. Siesel, “Visible Light-Harvesting in Covalently-Linked Porphyrin-Cyanine Dyes, Tetrahedron, 45, 4845, (1989); R. B. Mujumdar, L. A. Ernst, S. R. Mujumdar, and A. S. Waggoner, “Cyanine Dye Labelling Reagents Containing Isothiocyanate Groups”, Cytometry, 10, 11 (1989); L. A. Ernst, R. K. Gupta, R. B. Mujumdar, and A. S. Waggoner, “Cyanine Dye Labelling Reagents for sulphydryl Groups”, Cytometry, 10, 3, (1989); P. L. Southwick P. L., L. A. Ernst, E. W. Tauriello, S. R. Parker, R. B. Mujumdar, S. R. Mujumdar, H. A. Clever, and A. S. Waggoner, “Cyanine Dye Labelling Reagents-Carboxymethylindocyanine Succinimidyl Esters”, Cytometry 11, 418 (1990); R. B. Mujumdar, L. A. Ernst, Swati R. Mujumdar, C. J. Lewis, and A. S. Waggoner, “Cyanine Dye Labelling Reagents: Sulfoindocyanine Succinimidyl Esters”, Bioconjugate Chemistry, 4, 105, (1993); A. J. G. Mank, E. J. Molenaar, H. Lingeman, C. Goojer, U. A. Th. Brinkman, and N. H. Velthorst, “Visible Diode Laser Induced Fluorescence Detection in Liquid Chromatography after Precolumn Derivatisation of Thiols”, Anal. Chem., 65, 2197, (1993); H. Yu., J. Chao, D. Patek, S. R. Mujumdar, and A. S. Waggoner, “Cyanine dye dUTP analogs for enzymatic labelling of DNA Probes”, Nucl. Acids Res 22, 3226, (1994); Z. Zho, J. Chao, H. Yu, and A. S. Waggoner, “Directly labelled DNA probes using fluorescent nucleotides with different length linkers”, Nucl. Acids, Res, 22, 3226. A. J. G. Mank, H. T. C. van der Laan, H. Lingeman, Cees Goojer, U. A. Th. Brinkman, and N. H. Velthorst, “Visible Diode Laser-Induced Fluorescence Detection in Liquid Chromatography after Precolumn Derivatisation of Amines”, Anal. Chem., 67, 1742, (1995); S. R. Mujumdar, R. B. Mujumdar, C. M. Grant, and A. S. Waggoner, “Cyanine Labelling Reagents: sulfobenzoindocyanine succinimidyl esters”, Bioconjugate Chemistry, 7, 356, (1996). Patent Literature: P. L. Southwick, and A. S. Waggoner, “Intermediate for and Fluorescent Cyanine Dyes containing Carboxylic Acid Groups”, U.S. Pat. No. 4,981,977, Jan. 1, 1991; A. S. Waggoner, L. A. Ernst, and Mujumdar, R. B., “Method for Labelling and Detecting Materials Employing Arylsulfonate Cyanine Dyes”, U.S. Pat. No. 5,268,486, Dec. 7, 1993; A. S. Waggoner, “Cyanine Dyes as Labelling Reagents for Detection of Biological and Other Materials by Luminescence Methods”, U.S. Pat. No. 5,627,027, May 6, 1996; A. S. Waggoner, and R. B. Mujumdar, “Rigidised Trimethine Cyanine Dyes”, WO99/311181; G.-Y. Shen, T. S. Dobashi, “Cyanine Dye Activating Group with Improved Coupling Selectivity”; T. S. G. M. Little, R. Raghavachari; N. Narayanan; H. L. Osterman, “Fluorescent Cyanine Dyes”, U.S. Pat. No. 6,027,709, Feb. 22, 2000.
The general synthetic strategy necessary to prepare these labeling reagents is as follows. First, a quaternized nitrogen heterocycle Z1 is prepared. Then, this heterocyclic base is reacted with a polymethine linker (PML) that is an electrophilic reagent such as PhNH—(CH═CH)n—CH═NHPh.HCl or RO—(CH═CH)n—CH(OR)2, where Ph is a phenyl ring and R a methyl or ethyl group, to obtain a so-called hemicyanine dye, Z1—(CH═CH)nNHPh or Z1—(CH═CH)nNAcPh, where Ac is the acetyl radical, or Z1—(CH═CH)n—OR. These intermediates are then reacted with a different quaternary nitrogen heterocycle, Z2. The functionalized side arm can be attached either to the first or to the second quaternized nitrogen heterocycle.
The hemicyanine intermediates, however, can be difficult to obtain in good yields and/or in a pure form (see, for example, F. M. Hamer, “Some Unsymmetrical Pentamethincyanine Dyes and their Tetramethin Intermediates”, J. Chem. Soc., 32 (1949) and R. B. Mujumdar, L. A. Ernst, Swati R. Mujumdar, C. J. Lewis, and A. S. Waggoner, “Cyanine Dye Labelling Reagents: Sulfoindocyanine Succinimidyl Esters”, Bioconjugate Chemistry, 4, 105, (1993).
Thus polymethine dyes that are efficient and easy to produce as well as suitable for preparing conjugates with biomolecules are desirable.